JP7407987B2 - Proteomic assay using quantum sensors - Google Patents

Description

The present invention relates to the simultaneous detection of hundreds or thousands of proteins in biological fluids such as blood or urine in the first step of proteomic analysis. In particular, the invention uses a label-free assay that achieves such detection without using mass spectrometric or chromatographic methods and allows the use of equipment that is compact, inexpensive, and reusable. .

Traditionally, various attempts to assess gene activity or to decipher biological processes, including disease processes or pharmacological biological processes, have focused on genes. However, proteomics can provide further information about the biological functions of cells and organisms. Proteomics involves qualitative and quantitative measurements of gene activity by detecting and quantifying expression at the protein level rather than at the gene level. Proteomics also includes the study of non-genetically encoded events, such as post-translational modifications of proteins and interactions between proteins.

Currently, it is possible to obtain a large amount of genomic information. DNA chips are in practical use as molecular arrays for this purpose, and the cost of direct DNA sequencing continues to decrease significantly. Additionally, demand for high-throughput proteomics is increasing. Chips have been proposed to detect proteins, which are more complex and variable in biological function than DNA, which is currently the subject of intensive research for many applications. Proteomics is much preferred over genomics for health monitoring because the genome is static and represents only medical potential, whereas the proteome fluctuates dynamically with the patient's disease state and even This is because it is said to define those pathologies. However, it is difficult to detect and quantify proteins, whereas it is relatively easy to detect and quantify nucleic acids. This has motivated many attempts to measure mRNA (messenger RNA) concentration instead of protein concentration. Unfortunately, it has been shown that mRNA concentration does not correlate well with protein concentration. Proteomics appears to necessarily depend on the ability to directly detect proteins.

"Protein chip" is a collection used to refer to any device in which a protein or molecule for capturing the protein of interest (capture reagent) is immobilized on the surface of the chip, allowing detection of protein binding. This is a typical term. Until recently, capture reagents on protein chips were overwhelmingly antibodies. Detection of specific proteins in complex mixtures such as biological fluids requires high specificity, so many protein chips that utilize antibodies also rely on sandwich assays to boost specificity. Such assays are known to have important drawbacks for proteomics, some of which are specific to proteins and/or antibodies, some of which are specific to sandwich assays. .

Currently, there are no economically viable means by which proteomics data can be routinely collected from human subjects. Measurement of the proteomics of a sample is typically accomplished by various chromatographic techniques for sample preparation combined with various mass spectrometry techniques for detection and quantification. The equipment used in these techniques is expensive and large, so samples typically must be shipped to a central processing facility. The need to transport samples requires that the samples be processed at the time of collection for storage during shipping or stored in situ until transport is possible. Unfortunately, preparation and storage protocols tend to vary widely from location to location, and even within the same location. Differences in these protocols always result in significant variations downstream of proteomic measurements, making analysis difficult or impossible.

The ideal proteomics collection device would minimize costs by approaching fully solid-state operation (few moving parts) and utilizing label-free detection techniques such that reagent usage is minimal. limited, reusable for unlimited measurements, and operates with a small amount of biological fluid. Variations in sample analysis due to variations in sample preparation and storage protocols can be avoided by minimizing or eliminating sample preparation and performing sample measurements at predetermined locations and times and collections, eliminating sample storage and transportation. can be reduced by doing so.

A new class of non-protein-based capture reagents has been found with nucleic acid molecules. A long-standing theory has been that nucleic acids have a primarily informational role. Through a method known as SELEX (Systematic Evolution of Ligands by Exponential Enrichment), sometimes referred to as the SELEX process, it has been revealed that nucleic acids have three-dimensional structural diversity that is not significantly different from proteins. There is. The SELEX process is a method for the in vitro evolution of nucleic acid molecules that bind with high specificity to a target molecule, and is a method for the in vitro evolution of nucleic acid molecules that bind with high specificity to target molecules; U.S. Provisional Application No. 07/536,428 entitled "Systematic Evolution of Ligands by EXponential Enrichment," now abandoned U.S. Patent No. 5,475,096 (titled "Nucleic Acid Ligands") , US patent No. 5,270,163 (also see International Patent Publication No. 91/19813) (titled "Nucleic Acid Ligands"). Each of these publications, collectively referred to herein as a SELEX patent application, describes methods for making nucleic acid capture reagents for any desired target molecule.

The SELEX process provides a class of products called nucleic acid ligands or aptamers, each of which has a unique sequence and the property of binding specifically to a desired target compound or molecule. Each nucleic acid capture reagent identified by SELEX is a specific ligand for a given target compound or molecule. The SELEX process allows nucleic acids, whether monomers or polymers, to be used within their monomers to act as ligands (form specific binding pairs) with virtually any chemical compound. It is based on the unique knowledge that it has sufficient properties and sufficient chemical versatility to form a variety of two-dimensional and three-dimensional structures. Molecules of any size or composition can act as targets.

The SELEX method, which is suitable for high-affinity binding applications, uses the same general selection scheme to achieve virtually any desired criteria of binding affinity and selectivity from a mixture of candidate oligonucleotides. selection and stepwise iterations of binding, separation, and amplification. Starting with a nucleic acid, preferably a mixture of nucleic acids containing segments of randomized sequence, the SELEX method involves the steps of contacting the mixture with a target under conditions favorable for binding and binding specifically to the target molecule. separating unbound nucleic acids from the nucleic acids present, dissociating the nucleic acid-target complexes, and amplifying the dissociated nucleic acids from the nucleic acid-target complexes to yield a mixture of ligand-enriched nucleic acids; repeating the steps of binding, separating, dissociating, and amplifying through as many cycles as necessary to produce a high affinity nucleic acid ligand that is highly specific for the target molecule.

SOMAmers (Slow Off-rate Modified Aptamers) are aptamers with improved off-rate characteristics. This improvement in dissociation characteristics can be expressed as the dissociation rate (t _1/2 ) or the point at which 50% of the aptamer/target complex is dissociated. Such dissociation rates are generally 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, and 240 minutes. This is the average time for a protein-aptamer complex to dissociate. Additionally, SOMAmers contain modified nucleosides that provide different built-in functionalities. These functionalities can include tags for immobilization, labels for detection, means to facilitate or control separation, hydrophobic side chains to provide good affinity with proteins, and the like. A modification to improve affinity with proteins is a chemical group attached to the 5-position of the pyrimidine base. Functionalization of the 5-position (e.g., with a benzyl, naphthyl, or indole group) expands the chemical versatility of SOMAmer, allowing high-affinity binding to a broader range of target molecules. . Additionally, some polymerases can further transcribe DNA with modifications at these positions, thus allowing the amplification required for the SELEX process. SOMAmers and methods for making them are described in U.S. Pat. generating aptamers with improved off-rates).

Note that while aptamers and SOMAmers can be discovered by the SELEX process, other means for selecting them may exist. For example, as computational modeling of molecular interactions improves, ideal nucleic acid sequences for aptamers and relevant chemical modifications for SOMAmers can be directly calculated to create capture reagents specific for a given target molecule. may become possible. Other chemical techniques for screening aptamers and SOMAmers other than SELEX are also possible.

Assays aimed at the detection and quantification of physiologically important molecules in biological and other samples are important tools in the fields of scientific research and healthcare. Another class of such assays involves the use of microarrays containing one or more aptamers immobilized on a solid support. Each of these aptamers can each bind to a target molecule in a highly specific manner and with very high affinity. See, for example, U.S. Pat. (each entitled "Nucleic Acid Ligand Diagnostic Biochip"). These patents are herein incorporated by reference into this disclosure. When the microarray is contacted with a sample, the aptamers bind to each target molecule present in the sample, allowing the absence, presence, amount, and/or concentration of the target molecule in the sample to be determined.

Although label-free assays are considered highly desirable, they are not always achievable. A label is any exogenous molecule that is chemically or temporarily attached to a molecule of interest in order to detect the presence or activity of the molecule. Label-free assays utilize biophysical properties of molecules, such as molecular weight, molecular charge, dielectric constant, or (in the case of the present invention) affinity for aptamers, to monitor the presence or activity of molecules. do. Some embodiments of the invention utilize spin labels associated with aptamers attached to surfaces. Because this spin label is attached to a component of the detection system (eg, an aptamer) as opposed to a molecule of interest (eg, a protein), the present invention is considered a label-free assay. Many sensitive assays require that the analyte be "labeled" with a detectable tag. The tag or label can be a dye, a radioisotope, or anything else that is easily measured, by which the analyte can be measured by proxy.

Although ELISA assays (enzyme-linked immunosorbent assays) are considered the "gold standard" for immunoassays due to their high sensitivity and specificity, they are not considered label-free. ELISA uses two antibodies specific for different binding points (epitopes) on the analyte and is a "sandwich assay" as opposed to label-free. One of the antibodies is immobilized on the surface and captures the analyte from the sample fluid. The second antibody is linked to an enzyme that catalyzes a detectable change in the specific additive. After the analyte is captured by the immobilized antibody, this surface is washed to remove undesired molecules, and a second antibody is added, followed by washing and usually catalyzed addition to a detectable dye. Add agent. Although a second antibody confers specificity and detectability, it also requires several steps involving expensive reagents. The label-free form of this assay consists only of a first antibody and some means of detecting analyte binding.

Biochips, including protein chips, require a means to detect specific analytes in a sample while ignoring other molecules. The detection devices and methods described by the present technology are based on color centers located near the surface of a solid that can be probed via optical detection magnetic resonance (ODMR) or other techniques. Color centers are point defects near ideal transparent crystalline insulators or large bandgap semiconductors such as diamond, silicon carbide, or silica. These may consist of substitution defects, where an atom in the crystal is replaced by an atom of another type, vacancy defects, where no atom is present, or a combination of the two.

Color centers have localized electron orbitals that resemble free atoms. The electronic states are ordered in terms of principal, orbital and magnetic quantum numbers and can be stable if they are charged, neutral, or both. A wide bandgap or insulating crystal surrounding the color center acts as a "vacuum" separating the color center. At sufficiently low densities, this is an abundant and well-organized system with discrete optical transitions in the visible range (hence the term ``color centers'') that coexist with electron/nuclear spin states that have long relaxation times. yielding independent "atomic-like" entities with complex energy spectra that are split up.

Of particular interest are color centers whose fluorescence intensity depends on the spin polarization state (i.e., the ground state magnetic quantum number). In this case, the collection of magnetic sublevels is reflected in the fluorescence intensity, allowing optical detection of magnetic resonance. Since ODMR essentially converts radio frequency (RF) or microwave frequency detectables due to magnetic lower level transitions into the detection of much higher energy photons, it is superior in sensitivity (about 5 orders of magnitude) and It has clear advantages in terms of strong fluorescence, allowing optical observation of individual color centers.

An example of a color center capable of such ODMR is the nitrogen-vacancy (NV) in a diamond crystal. Although the preferred embodiment described in this invention utilizes NV centers in diamond for detection, the invention can also use other color centers. Diamond itself has over 500 known color centers, the majority related to nitrogen. Other elements known for possible substitution in the diamond lattice include nickel, boron, silicon, hydrogen, and cobalt. Color centers in other crystal lattices include, for example, germanium-related defects in germanosilicate glasses, silicon defects in silicon carbide, and X-ray induced defects in _LiBaF3 crystals.

As the name suggests, NV centers consist of nitrogen substitutions for carbon atoms located next to adjacent vacancies in the lattice. This is illustrated in FIG. 1, which is a schematic diagram of a diamond crystal, indicated generally at 100 and including an NV center indicated at 104. NV centers are paramagnetic color centers with an inherent coupling between their electronic spin states and optical states. It can emit strong and stable fluorescence (i.e. large absorption coefficient combined with short-lived excited states), also exhibits very long magnetic relaxation times, and is sensitive to local properties such as magnetic or electric fields. It becomes a detector. Diamond itself is extremely stable mechanically, thermally, and chemically. At the same time, the diamond surface is suitable for chemical modification, which is useful for binding molecular agents. Pure diamond is optically transparent, allowing free excitation and emission of fluorescent centers; this fluorescence also exhibits no photobleaching and minimal luminescence intermittency (“blinking”). The combination of all these properties makes NV centers good candidates for highly sensitive biosensing.

The present disclosure provides methods for detecting target molecules in a sample based on changes in the properties of one or more color sensors placed near the surface of a substrate upon binding of the target molecules to a capture reagent attached to the surface. Apparatus, systems, and methods are described. The detector may be configured to detect target molecules in the sample by detecting changes in the properties of the color center. Optionally, the target molecule is a protein and the capture reagent is an aptamer. In some cases, the color center is a NV center located in the diamond and the change in property is a change in fluorescence emission. In some cases, large numbers, eg, hundreds, thousands, tens of thousands, or even more, of protein species can be targeted in one assay.

The technology involves attachment of an aptamer or SOMAmer on a surface, such as a diamond surface, and passivation of areas of the surface that do not bind the aptamer or SOMAmer against non-specific binding by unwanted proteins. The technology also involves the regeneration of surface-attached aptamers or SOMAmers for several additional rounds of protein detection.

This technique aims to eliminate all moving parts except those related to the delivery of bulk fluids to the active surface of the biochip. These fluids include sample fluids, wash fluids, and fluids used for biochip regeneration. The proposed scheme for detection is a label-free method, meaning that no detection agent needs to be added to the sample to enable measurement of the protein of interest. Regeneration of the active surface is non-destructive and requires only mild buffer washes to dissociate proteins from aptamers or SOMAmers. The NV center is extremely stable and the aptamer and SOMAmer itself are extremely stable and can be used hundreds of times.

More specifically, after the sample liquid contacts the active surface, some time is required for the proteins to bind to the immobilized aptamer. This binding time also includes the time required for the protein to diffuse from the bulk liquid to the surface, which can take several hours. During this binding time, the sample fluid can be static, but it is more common to agitate or recirculate the fluid to bring unbound proteins closer to the surface, thereby reducing the distance they need to spread. be. After the binding step is completed, it is common to wash the surface to remove unbound protein. This washing is generally performed using a buffer solution similar to the sample liquid, such as phosphate buffer, or distilled water. If highly specific detection is required, washing can be performed by using high salt concentrations, mild denaturing agents such as low concentrations (<1 M) of urea, or by including competitive agents for the protein of interest. It can be further strengthened. Such competitive agents may be common, for example albumin, which competes with proteins, or heparin, which competes with nucleic acids. Nucleic acids in various bulk forms can also be used, such as salmon sperm DNA, or random DNA synthesized in bulk. Competitors may also be unique, eg, the use of proteins similar to the protein of interest, or non-human proteins that compete with the human protein. Regarding binding, the wash liquid may be static or agitated, requiring some time for diffusion from the surface into the bulk liquid.

This technology eliminates most discrepancies in sample analysis by performing the assay and analysis at the point of collection, such as inside the toilet being tested. Assaying and analyzing fresh urine in a collection container (i.e., toilet) eliminates the need to store and transport samples, as well as the associated inconsistency.

This ability to perform routine proteomics inexpensively at the consumer level will have profound implications for science and healthcare. Proteomics science is lagging far behind its potential due to the simple lack of large numbers of good quality samples required for meaningful analysis. The present technology significantly facilitates both the collection and analysis of such samples. Good proteomics science yields good medical diagnostic predictions. However, such diagnostic predictions are of little practical use in health care if patient-derived samples are not easily collected and analyzed. Thus, providing good proteomics science, this technology will also provide easy use in healthcare science.

Various other features of systems and methods according to the present technology are described in this disclosure. The properties, features, and advantages may be achieved independently in various embodiments of the present disclosure or may be combined in further other embodiments, further details of which may be found with reference to the following description and drawings. It can be understood.

FIG. 1 is a schematic diagram showing nitrogen-vacancy centers in diamond. FIG. 2 is a schematic diagram of target molecule detection according to aspects of the present technology. FIG. 3A is a schematic illustration of an interaction mechanism that may be used to detect target molecules, according to aspects of the present technology. FIG. 3B is a schematic illustration of another interaction mechanism that may be used to detect target molecules, according to aspects of the present technology. FIG. 4 is a schematic illustration of electron energy levels at diamond nitrogen vacancy (NV) centers and transitions between these levels, in accordance with aspects of the present technology. FIG. 5 depicts exemplary optical absorption and emission spectra of NV centers in accordance with aspects of the present technology. FIG. 6 is a schematic illustration of optical readout and spin polarization of an NV center in accordance with aspects of the present technology. FIG. 7A is a schematic illustration of excitation and emission pulses associated with an NV center, according to aspects of the present technology. FIG. 7B is a graphical representation of NV-centered emission intensity comparing intensity as a function of time in the absence and presence of bound target molecules, according to embodiments of the present technology. FIG. 8A depicts a first exemplary configuration of an apparatus for detecting a target molecule, according to aspects of the present technology. FIG. 8B depicts another exemplary configuration of an apparatus for detecting a target molecule, according to aspects of the present technology. FIG. 8C depicts yet another exemplary configuration of a device for detecting a target molecule, according to aspects of the present technology. FIG. 8D depicts yet another exemplary configuration of an apparatus for detecting a target molecule, according to aspects of the present technology. FIG. 9 depicts yet another exemplary configuration of an apparatus for detecting a target molecule, according to aspects of the present technology. FIG. 10 is a flowchart depicting exemplary steps in a method of detecting a target molecule, according to aspects of the present technology. FIG. 11 is a flowchart depicting exemplary steps in another method of detecting a target molecule, according to aspects of the present technology. FIG. 12 is a flowchart depicting exemplary steps in yet another method of detecting a target molecule, according to aspects of the present technology. FIG. 13 is a flowchart depicting exemplary steps in a method of manufacturing a device for detecting a target molecule, according to aspects of the present technology.

FIG. 1 is a schematic diagram showing nitrogen-vacancy centers in diamond. In its pure form, diamond crystal 100 consists only of carbon atoms 101. When nitrogen atoms 102 replace carbon atoms in the diamond crystal and are placed adjacent to vacancies 103 in the crystal, nitrogen-vacancy centers 104 are created.

の方向である。さらに、いずれかの所定の方向において、窒素置換および空孔のオーダーは反転し得、よって、実際には８つの固有のＮＶ中心の構成が存在する。 As can be seen in Figure 1, each carbon has four identical binding partners, so there are four crystals in which the axis of the NV center can be located depending on how the vacancies are arranged in relation to the nitrogen substitution. There is a scientific direction. these are,

The direction is Furthermore, in any given direction, the order of nitrogen substitution and vacancies may be reversed, so there are actually eight unique NV center configurations.

The interaction of a molecule with the NV center depends not only on its distance to the NV center, but also on the relative orientation of the two. Additionally, there are optical directions for the excitation and detected light. All of these considerations must be taken into account when choosing which face of the diamond crystal is used as the detection face.

Nitrogen-vacancy centers are created by introducing nitrogen impurities and vacancies at a predetermined depth, then aligning the vacancies via diffusion with respect to the nitrogen impurities, and annealing at 1000K to 1300K. Can be embedded at depth into crystal structures such as diamond. Nitrogen defects can be implanted at the desired depth via nitrogen pulses during chemical vapor deposition (CVD) of the diamond matrix or by ion beam implantation after deposition is complete. Holes can be implanted or created via e-[54], H+[55] or He+ ion beams.

Natural diamond contains about 1% ^13C , which has 1/2 the nuclear spin (which is the paramagnetic nature of the nucleus) and therefore interacts with the NV center, so the relaxation time of the NV center is less than CVD. can be augmented by growing a deposited layer of isotopically pure ¹² C diamond on an existing crystal and creating NV centers within this layer. Similarly, 1/2 nuclear spin of ¹⁵ N is preferred over 1 nuclear spin of ¹⁴ N isotope. Also, since nitrogen substitution carries spin, the greater the percentage of nitrogen that can be converted into NV centers, the more transparent the sample will be from a magnetic point of view.

Under appropriate CVD conditions, the orientation of the created NV centers can be significantly biased. That is, rather than all eight possible orientations being equally populated, a single orientation may occur preferentially over others. Alignments as high as 99% may be possible. This can be very useful for creating devices where all of the NV centers are identical and can be optimally oriented. For example, if the sensing surface of diamond is a (111) plane and the majority of NV centers can be oriented along the [111] direction, they can all be oriented perpendicular to the sensing surface.

In some embodiments of the present technology, it is desirable to create a very thin layer of diamond deposited on another substrate, such as silicon, where additional detection electronics are present. Another use for ion implantation is to insert a "break layer" at a certain depth into a thick diamond crystal using, for example, hydrogen atoms. After bonding thick diamond crystals to other substrates, the diamond may be mechanically impacted and will break along the fracture layer, leaving a thin layer of diamond crystals. Similar technology is already used in the semiconductor industry.

FIG. 2 is a schematic diagram of target molecule detection according to aspects of the present technology. A capture reagent 200 is deposited on a surface 202 of a substrate 203 (eg, a crystalline film, a diamond film, and/or a high purity insulator such as single crystal diamond) in close proximity to a color center 204 . When the color center is illuminated with excitation light 208 (eg, from light source 209), it is stimulated to emit fluorescence 210, which is characterized by spectrum and intensity. Capture reagent 200 is contacted with sample liquid 211 (e.g., by exposing surface 202 to the sample liquid) such that target analytes 206 (also referred to as target molecules) in the sample solution bind to capture reagent 200. A complex 212 is formed, thereby resulting in and/or altering an interaction 214 with the color center, which results in a detectable change in the nature (eg, intensity) of the emitted fluorescence 216. In interaction 214, the color center may detect a change in the electric field or a change in the magnetic field upon binding of the target molecule to the capture reagent. Properties of the color center (eg, magnetic resonance or spin related properties of the color center) can be altered by interactions 214.

The sample fluid can be a biological fluid. Capture reagent 200 can be a nucleic acid molecule, oligonucleotide, aptamer, SOMAmer, or any other binding agent configured to bind to a target molecule. In some examples, the capture reagent comprises a pyrimidine modified at least at the 5-position (ie, a pyrimidine modified at the 5-position, such as uridine or cytidine). In some examples, different types of pyrimidines modified at the 5-position are attached to the surface. For example, at least one 5-position modified uridine and at least one 5-position modified cytidine can be attached to the surface.

The target analyte can be a protein. Proteins include normally folded polypeptide chains, abnormally folded polypeptide chains, unfolded polypeptide chains, fragments of polypeptide chains that may or may not be normally folded, and short polypeptide chains. , polypeptides that incorporate unnatural amino acids, and polypeptides that are post-translationally modified (eg, phosphorylated, glycosylated, disulfide bonds, etc.) or incorporated into protein complexes. Target analytes may also include small molecules found in biological fluids such as metabolites.

Figures 3A-3B are schematic illustrations of two potential interaction mechanisms for detection techniques, where the color center is the NV center, the target analyte is a protein, and the capture reagent is an aptamer.

FIG. 3A is a schematic illustration of an interaction mechanism that may be used to detect target molecules utilizing magnetic spin labels, according to aspects of the present technology. Stable organic molecules are usually diamagnetic, and paramagnetic molecules (i.e. "free radicals") have unpaired electrons and are therefore chemically active and can be Tends to lose spin. Stable free radicals that escape exposure to the biochemical environment are exceptional. Spin labels are stable paramagnetic organic molecules or complexes that can bind to another molecule, especially nucleic acids and amino acids, developed specifically for site-specific spin labeling of large biomolecules.

The most commonly used spin labels are small organic molecules containing nitrogen oxides, developed for incorporation into nucleic acids, or metal chelators such as EDTA, which complex with high affinity for paramagnetic metal ions. Nitric oxide derivatives of nucleobases have also been developed. In recent years, triarylmethyl (trityl) radical derivatives have also become popular for labeling both proteins and nucleic acids. EDTA derivatives of both deoxyribo-thymine and deoxyribocytosine have been developed in the form of phosphoramidites for use in DNA synthesizers. For measurements where the spin label is intended to simply change the relaxation time of the NV center, it is important that magnetic interactions exist and that a wide variety of spin labels are applicable. However, in measurements where the spin labels of the labels are directly presented, the individual spectra of the spin labels become related. For example, in DEER, the narrower the spectrum and the longer the spin label lifetime, the better. As a specific example, triplet splitting of the spectrum is disadvantageous due to hyperfine coupling to ¹⁴ N in DEER, and a single line spectrum of deuterated trityl is the preferred choice.

In FIG. 3A, an aptamer 301 coupled to a magnetic spin label 300 is attached to the sensing surface 202 of the diamond crystal 100 in close proximity to the color center 104. The target protein 302 in the sample solution binds to the aptamer to form a complex 303, which affects the interaction 304 between the spin label and the NV center by changing the proximal relationship of the NV center and the spin label. This results in a detectable change in the nature of the fluorescence of the NV center.

More specifically, FIG. 3A illustrates a particular configuration designed to maximize magnetic interactions by placing spin-labeled aptamers close to NV centers in diamond. Conformational changes upon protein binding can shift the spin label in relation to the NV center. Movement closer to the NV center increases the magnetic field at the NV center and results in measurable changes in the dynamic or quasi-static features of the lower level spectra of the NV center.

The lower level spectrum may be probed using a detector assembly configured to illuminate the NV center with excitation light and detect emission of electromagnetic radiation from the NV center. The detector assembly detects changes in the spectrum of the lower level by illuminating the NV center with microwave radiation over a range of frequencies including the resonant frequency of the lower level and detecting changes in the radiation emitted by the NV center. It is possible. The radiation at the resonant frequency may induce a conversion of the ground state electrons of the NV center from the first sublevel to the second sublevel, thereby inducing a change in the characteristics of the emitted radiation. An example of a characteristic of the emitted radiation is the relationship between the frequency of the excitation light and the frequency of the emitted light. Furthermore, adding spin labels along with microwave excitation opens a wide range of options for manipulating spin label interactions with NV centers.

FIG. 3B is a schematic illustration of another interaction mechanism that may be used to detect target molecules using electrostatic interactions, according to embodiments of the present technology. Aptamer 301 is attached to sensing surface 202 of diamond crystal 100 in close proximity to NV center 104 . Both cations 308 and anions 309 are present in the fluid being analyzed. The negatively charged backbone of the aptamer attracts cations 308. These bound ions influence the local electric field relative to the NV center such that when the NV center is illuminated with excitation light 208, it emits fluorescence 310 characterized by its spectrum and intensity. stimulated. Proteins in the sample solution also have ions bound to them due to their charge. This protein binds to the aptamer to form a complex 303, thereby altering the charge distribution and thus the local electric field relative to the NV center, resulting in a detectable change in the nature of the emitted fluorescence 312. .

More specifically, FIG. 3B illustrates a configuration in which electrostatic interactions may be explored. The presence of mobile charges near the surface significantly affects the relaxation time of the NV centers. There is also a negative charge attached to the NV center itself and a negative charge attached to the backbone of the aptamer. Additionally, the target protein itself can be charged. These charges are screened by counterions in solution. Binding of a protein molecule to the aptamer rearranges this charge distribution relative to the NV center. Furthermore, protein molecules themselves have a significantly lower dielectric constant than water, and their presence affects the screening of charge distribution. Both effects result in changes in the quasi-static and dynamic electric field present at the NV center, affecting lower-level spectral features.

The degree to which static or dynamic changes in the lower-level spectrum dominate the interaction can be influenced by the environment in which the measurements are made. If measurements are made at room temperature, charge mobility in the solution and the molecule itself is expected to contribute to significant dynamic changes that can be characterized by direct measurements of _T1 . In contrast, if measurements are made at temperatures below the freezing point of the biological fluid, these dynamic changes are suppressed, allowing direct detection of static disruption at lower levels. For spin-labeled aptamers, performing measurements in the frozen state significantly increases the relaxation time of the spin-label and can be used to directly measure the change in distance between the spin-label and the NV center. It enables technologies such as DEER (electron-electron double resonance).

The optimal configuration for locating the NV center also depends on whether the spectral changes are dominated by quasi-static or dynamic changes. If dynamic changes are dominant and direct T ₁ measurements are used, it is possible that the NV centers are located closer to the surface and can be located closer to each other. On the other hand, careful measurement of quasi-static changes to the spectrum requires longer relaxation times (first T ₁ ) with high fidelity and thus further away from the surface and well separated from each other. Requires NV center. The distance of the NV center from the surface is between maximizing the interaction of the aptamer and its bound target and keeping long relaxation times creating NV-sensitive detectors of quasi-static electric and magnetic fields. It is a compromise plan.

Figures 3A-3B investigated two limits of interaction, where magnetic and electric fields are the dominant ones that lead to changes in the NV center upon binding of the analyte. Similar to when the SELEX process was modified to screen for slow dissociation of SOMAmer production, candidate aptamers can be coupled to a device such as the one described herein and the NV-centered Steps to measure responses may be added to subsequent selection rounds. In this way, SOMAmers that maximize signal contrast during binding events can be maximized.

FIG. 4 is a schematic illustration of the electron energy levels of nitrogen vacancy (NV) centers in diamond and the transitions between these levels, in accordance with aspects of the present technology. More specifically, FIG. 4 is a simplified schematic of the electron energy levels of negatively charged NV centers in diamond and the transitions between these levels. The ground state 399 of the electron at the NV center includes three sublevels (also called substates or states). The zero spin lower level 400 has the lowest energy and is denoted | ³ A ₂ ,0>. The lower level of spins 402 with m _s =+1 and m _s =−1 have the same energy in the absence of an external field and are denoted | ³ A ₂ ,±1>.

The external field affects the spin state with m _s = ±1, but not the spin state with m _s =0, lowers the energy of the spin state with m _s = -1, and lowers the energy of the spin state with m _s = +1. Increases the energy of the spin state. For example, a 1027 Gauss magnetic field aligned with the symmetry axis of the NV center reduces the level of | ³ A ₂ ,−1> to the level of | ³ A ₂ ,0> at room temperature. The presence of magnetic spin labels in the immediate vicinity of the NV center has a similar but significantly smaller effect. Irradiation with microwaves at a modified frequency 404 (eg, the resonant frequency) induces a transition from the base m _s =0 spin state to the base m _s =±1 spin state. A transition 406 from a ground m _{s =0 spin state to an excited m s} =0 spin state, denoted | 3 E,0>, or a transition 406 from the ground m _s =0 spin state ^, denoted | ³ E,±1> A transition 408 from the m _s =±1 spin state of the ms = ±1 spin state to the excited m _s =±1 spin state 408 may be induced by irradiation with excitation light at the modified frequency 208 .

The transition from the excited state to the corresponding ground state is accomplished by the emission of a red photon 418. Alternatively, the transition from the excited state to the ground state may be a so-called "dark transition" since most or all the transition is achieved without any emission of photons or only with the emission 412 of longer wavelength infrared photons. transition) 410. The dark transition 410 can be achieved with the emission of photons 412 in the infrared range that are absorbed by the diamond lattice, or along a path 414 with no photon emission at all. In most cases, the dark transition results in a spin transition 416 from | ³ E,±1 to | ³ A _2,0 >. Approximately 30% of the electrons on transitions at the | ^3E ,±1> level transition to the ground state via a dark transition, resulting in the emission of a detectable photon. Therefore, an electron in a spin state of m _s =±1 provides only about 70% of the fluorescence of an electron in a spin state of m _s =0.

Even more specifically, FIG. 4 shows a schematic energy level structure consistent with findings and calculations for negatively charged NV centers. Six electrons are located in the ground state "molecular orbitals" of _C3V symmetry (imposed by the static lattice structure). These trajectories are linear combinations of four trajectories that are consistent with calculations of diamond's band structure. Energistically, this ground state, labeled ³ A ₂ in Figure 3, is a deep level state in the bandgap. The first excited state, labeled ^3E , is 1.94 eV above the ground state. If it is less localized, it will be further away from the conductor by more than 1 eV. The 1.94 eV difference between the two band states corresponds to the observed 638 nm zero phonon absorption and emission lines shown in FIG.

Both the ground state and the excited state are spin triplets with S=1, but hereafter we will focus only on the magnetic sublevels of the ground state. Thus, rather than using the intractable state description in Figure 3, we often simply refer to the state where m _s =0, or m _s =±1, which we is understood to refer to the ground state.

In the absence of an applied field, the lower level degeneracy of m _s =0 and m _s =±1 in the ground state is due to large zero field splitting (2.88 GHz) along the NV central axis. The first is enhanced, reflecting the strongly non-spherical wavefunction of the six electrons. In the presence of an applied magnetic and/or electric field, the lower levels of m _s =0 and m _s =±1 are further split. For fields parallel to the NV central axis, the splitting is 2.8 MHz per Gauss in the magnetic field and 3.5 mHz per V/m in the electric field. This figure does not represent the energy differences to scale; the magnetic lower level splitting is about 5 orders of magnitude smaller than the main splitting between the ground and excited states (1.95 eV).

Transitions between ground state lower levels 400 and 402 and excited states 406 and 408 are present in the visible range, resulting in interesting photoluminescence properties of the NV centers. FIG. 5 depicts exemplary spectra of light absorption and emission of NV centers at room temperature, in accordance with aspects of the present technology. The absorption spectrum 500 peaks at about 570 nm and shows how efficiently photons of a given wavelength are absorbed by the NV center. Emission spectrum 502 peaks at approximately 690 nm and indicates the relative intensity of light emitted at a given wavelength. Absorption of a photon of any wavelength in the absorption spectrum can result in emission of a photon of any wavelength in the emission spectrum. The expected spread of the spectrum from different wavelengths is due to the dissipation of vibrational energy into the molecular lattice or the absorption of thermal energy from the molecular lattice. Both spectra share a peak 504 where the absorbed and emitted photons have the same energy. This peak is called the zero phonon line, where these spectra converge as the atmospheric temperature reaches absolute zero and the vibrations of the lattice are frozen.

More specifically, both the absorption and emission spectra exhibit strong absorption coefficients, short (about 4 ns) lifetimes for the excited state, and wide phonon broadening resulting in strong fluorescence with virtually no bleaching. Direct absorption and emission paths leave both S and m _s uncharged (the angular moment of the absorbed or emitted photon is canceled by the change in the momentum of the "molecule's" orbit) ). In contrast, strong phonon coupling also allows another decay path (via intermediate system crossing), which changes m _s by one, as shown on the right side of Fig. 3. This decay path goes through multiple intermediate states and the energy of any photon emitted is further in the infrared compared to the photon provided by the direct path. Thus, this is often referred to as the "dark path" and is higher for electrons excited from the lower level of m _s =±1 than for those excited from the lower level of m _s =0. Hired based on potential. The net result for electrons excited from the lower level of the ground state m _s =0 is a higher visible fluorescence intensity compared to those excited from the lower level of m _s =±1.

FIG. 6 is a schematic illustration of optical readout and spin polarization of an NV center in accordance with aspects of the present technology. An excitation pulse 600 illuminates the nitrogen-vacancy center and stimulates the emission 602 of the pulse. The initial intensity 604 of the emitted pulse, I _start , is the minimum point of the pulse and represents the relative set of states m _s =0 and m _s =±1 at the beginning of the measurement. This intensity quickly increases to a maximum intensity 606, I _max , as the spin state becomes polarized with respect to the m _s =0 state.

The change in the magnetic sub-level ensemble is exactly what is observed via electron spin resonance (EPR), where the frequency of the applied microwave excitation corresponds to the sub-level splitting in m _s . The fluorescence intensity depends on the above-mentioned sub-level assembly and allows optical detection of ground state magnetic resonance (ODMR) above the NV center. Although Applicants continue to refer to ODMR detection, it is noted that direct electrical detection of the photocurrent at the NV center is illustrated and can serve as another detection mechanism.

Conventional magnetic resonance applies continuous or pulsed microwave/RF electromagnetic fields to simultaneously induce transitions between or mix the magnetic sublevels of a large number (>10 ¹³ ) of spins. , that is, it operates on a macroscopic (ensemble average) magnetization that depends on temperature. At zero absolute temperature, all spins are at their lowest energy sublevel and the magnetization of the system is maximum, so this state is 100% spin polarized. At finite temperature, lower-level ensembles follow Boltzmann statistics, decreasing the polarization of the ensemble mean as temperature increases. At room temperature, the energy difference between sublevels is so small that a collection of different magnetic sublevels is roughly equal, resulting in only 0.1% polarization.

The magnetization that arises from the differences in the lower level sets is called longitudinal magnetization, or the z-component of the magnetization since the z-axis is conventionally chosen parallel to the quantization axis. At any temperature, longitudinal magnetization has an equilibrium value that is proportional to the spin polarization. Transverse magnetization, which is the XY component of magnetization, is an ensemble average of mixed states (eigenstates are located along the Z direction). Its equilibrium value is zero.

Lower-level transitions or mixed states (with a non-zero ensemble mean of transient transverse magnetization) can be created by applying an RF/microwave electromagnetic field with a photon energy that matches the energy of lower-level splitting, E. . The corresponding frequency is defined by E=hf _L , where h is Planck's constant and f _L is known as the Larmor frequency. The mixed state (and thus the ensemble average transverse magnetization) precesses (rotates) at the Larmor frequency. Typically, the EMF induced by this rotating macroscopic magnetization is detected via a coil or resonator device.

After the creation of a mixed state, the transverse magnetization signal essentially decays over time for two reasons:
1. The individual spins experience different or changing fields during precession, i.e. the lower level energy differences in m _s are different, so that the precession frequencies of the mixed state are slightly different. . While they start precessing in phase, the phase difference builds up due to the precession frequency difference and the ensemble average (vector sum) transverse magnetization progresses to zero. Since the precessing macroscopic magnetic moment collapses to zero, the necessarily induced signal also collapses. The characteristic time of this collapse, caused by the loss of “phase coherence”, is called _T2 or the transverse relaxation time. In more detailed experiments, decays due to static field differences (i.e. magnetic field inhomogeneities across the sample) are eliminated, and decay times that reflect only dynamic changes during the measurement are therefore reduced to phase memory times. time) _TM . This form of loss of transverse magnetization does not require energy exchange with the environment.
2. At finite temperature, spins can exchange energy with the environment and flip from one sublevel to another, ie, the eigenstates have a finite lifetime due to their interaction with the environment. More precisely, the fluid (time-dependent) electromagnetic field introduced by the environment needs to have a transverse non-zero Larmor frequency field component to induce a change of state. As a result of these changes, the precessing transverse magnetization is lost as the magnetization returns to the z (or longitudinal) direction. The characteristic time associated with this process (the time required to return to the thermally equilibrium collective distribution of eigenstates) is called T ₁ or longitudinal relaxation, or, since it requires an exchange of energy with the environment, the spin - called lattice relaxation time. This process imposes an upper limit on any precession decay times, such as T ₂ and T _m .

Unlike conventional EPR detection, ODMR detects longitudinal magnetization. However, the loss of lateral and its advantages is markedly compensated for by a significant increase in susceptibility: low-energy magnetic lower-level transitions can generate photons with almost perfect quantum efficiency and more than 1000,000 times more energy. can be observed through.

In addition to allowing ODMR detection, in isolated NV centers, _ms -dependent fluorescence also allows manipulation of lower-level assembly. This is because the lifetime of magnetic sublevels is much longer than that of fluorescence. At sufficiently high excitation intensities, the fluorescence rate is limited only by the lifetime of the fluorescence and can be 100 MHz or higher. For example, an excitation from a lower level at m _s =±1 of the ground state has a probability of w = 0.7 to retain its quantum number and a probability of 0.3 to return via a dark path to a lower level at m _s =0. , the probability of finding any of the spins in the state m _s =0 after 10 cycles is 98%, i.e. 98% spin polarization can be constructed for the macroscopic sample. Since T ₁ at the NV center is typically much longer than 100 microseconds, a sufficiently strong 100 ns pulse can create spin polarization equivalent to cooling the diamond from room temperature to 0.3 K. Starting any magnetic resonance measurement with nearly perfect spin polarization is a 100-fold increase in signal compared to starting with equilibrium spin polarization at room temperature.

The same pulse that polarizes a collection of spins also acts as a measurement of spin polarization, as shown in FIG. Maximum fluorescence occurs when the sample is 100% spin polarized at levels below m _s =0. Applicants define the maximum fluorescence intensity as I _max . If we use the probabilities from the example above and assume that we are starting at thermal equilibrium, when the excitation light is turned on at t=0, the fluorescence response is 1/3 of the lower level set with _ms = 0 which fluoresces with maximum efficiency, and _ms = ± which fluoresces with 70% efficiency (1/3 + 2/3 * 0.7 = 0.8) 80% of I _max , corresponding to 2/3 of the state of 1. This intensity reaches I _max asymptotically at a ratio determined by the intensity of the excitation light.

として、最初の蛍光強度Ｉ_{ｓｔａｒｔ}の観点から表され得る。 In this case, the initial polarization of the lower level with m _s =0, P ₀ , is

can be expressed in terms of the initial fluorescence intensity I _start as .

In the case of w=0.7 analyzed above, if I _start =I _max then P ₀ =1. If I _start =0.7*I _max then P ₀ =0. If I _start =0.8*I _max then P ₀ =1/3, and so on.

The unique properties of NV centers summarized above mean that it is easy to construct an experimental setup that can detect the fluorescence of a single NV center using commercially available fluorescence microscopes. Because NV centers can be separated by hundreds of nanometers, the fluorescence of a single NV center observed under a microscope encompasses all the spectroscopic information at the magnetic sublevels of a single spin. When a single spin is observed, there is no ensemble average as in the case of conventional EPR measurements, but there is still a direct correspondence between the ensemble average and the time average of a single spin. To relate these two, the value of the ensemble mean polarization needs to be replaced by a lower-level _probability , i.e., the optical The excitation pulse is translated to find a single NV center with _ms = 0 state with 98% probability. Thanks to high fluorescence, rapid repetition and sufficient time averaging, reasonably short experimental times with low statistical errors can be achieved.

The properties of NV centers are well suited for time-domain magnetic resonance: 100% polarized initial state, sensitive detection of single centers, and long relaxation times, as well as sophisticated high-resolution spectroscopy. The fact that all time-domain RF/microwave pulse sequences invented to date can be applied makes the single NV center the most sensitive quantum measurement tool available.

Figures 7A-7B represent a means for measuring the relaxation time, T ₁ , through entirely optical measurements. The lower level of spins with m _s = ±1 emits with approximately 70% fluorescence intensity of the lower level with m _s =0, so that if divided evenly, the total fluorescence intensity is the same as the spins with m _s = It is about 80% of the case when it is completely polarized to a level below zero. A series of pulses as in Fig. 6 can be used to evaluate the relative set of lower levels of m _s =0 and m _s =±1, as well as to repolarize the spins to the lower level of m _s =0. enable measurements to be made.

FIG. 7A is a schematic illustration of optical excitation pulses 708 and emission pulses 710 associated with an NV center, according to aspects of the present technology. The NV center in the diamond is irradiated with a series of pulses of excitation light, each of sufficient duration to result in complete polarization of the NV center to the lower level of m _s =0. The spacing τ _i between these excitation pulses varies. The fluorescence intensity just after the start of the excitation pulse (filled squares I ₀ , I ₁ ...) is taken when the spin state should be fully polarized at the lower level of m _s =0. , as well as the reference intensities (open rectangles) are measured.

FIG. 7B is a graphical representation of NV center emission intensity comparing intensity as a function of time in the absence and presence of bound target molecules, according to embodiments of the present technology. More specifically, in Fig. 7A the difference between the initial and reference intensities is plotted as a function of the time interval τ _i between them, and how long it takes for the lower level to return to thermal equilibrium. indicates how much time is required. An exponential fit of intensity=I ₀ *exp(−τ/T ₁ ) yields a direct measurement of T ₁ . Data points 700 indicate a more rapid return to equilibrium, so their exponential fit 702 yields a shorter T ₁ than data points 704 and their exponential fit 706. Different T ₁ values result from analyte bound or unbound to the aptamer.

More specifically, in FIGS. 7A-7B, a series of excitation pulses of sufficient duration and intensity to optically polarize the NV center are applied with varying waiting times therebetween. This excitation wavelength is shorter than the zero phonon line; in practice, 532 nm is often used. While the excitation pulse is applied, emission from the NV center is monitored in a bandwidth that includes part or all of the emission spectrum 502.

Initially, the ground state is thermally equilibrated and thus the initial level of fluorescence _I0 is less than the maximum possible _Im since the states with m _s = ±1 and m _s =0 are approximately equally present. correspond to the level. Over the course of the first pulse, the emission grows to a maximum value and saturates at I _m as the state m _s =0 comes into existence due to the dynamic spin polarization process described above. After a waiting period, a second pulse is applied. If the waiting time is short enough, the interaction with the surrounding environment will not have enough time to equilibrate the ground state population and there will still be many m _s = 0, thus reducing the first emission measured. The intensity I ₁ is still close to the maximum value I _m .

Spin polarization occurs again during the remaining second pulse, and the state is again polarized to m _s =0 and a corresponding intensity of I _m . A longer waiting period then follows and the whole process is repeated again. This process is eventually repeated so that the waiting time is substantially longer than the relaxation time of the NV center, the ground state is thermally equilibrated, and all three sublevels are equally present. , the first emission measured is again equal to I ₀ .

By plotting the initial emission intensities I ₁ , I ₂ , I ₃ . . . as a function of waiting time, a graph such as that in FIG. 7B can be created. The fit to the graph may yield the NV-centered relaxation time _T1 . Figure 7B shows examples of two data sets and their respective fits corresponding to different _T1 values. This measurement sequence is very similar to that used in standard pulsed magnetic EPR experiments, where the initial state is an inverted thermal equilibrium magnetization prepared by microwave π pulses - M ₀ , and the later time value is read by detecting the microwave radiation initiated by another microwave π/2 pulse. However, in this case no magnetic field or microwave antenna or resonator is required, which considerably simplifies the measurements.

In addition to microwaves, more detailed measurement techniques become possible. For example, for the fully optical _T1 measurements described above, every polarizing light pulse is immediately followed by an added microwave π-pulse (with a frequency of zero field splitting and oriented perpendicular to the symmetry axis of the NV center). ) provides an initial population of zeros for the state m _s =0. By alternating measurements with and without microwave pulses and plotting and fitting their differences, T ₁ can be determined without the need for fitting the values of the remaining (thermal equilibrium) signal. Note that this works very simply because the state with m _s =+/−1 in the absence of an external magnetic field is degenerate (equal energy).

Adding an external field further increases measurement possibilities. When an external static field H ₀ is imposed, the energy level of the state m _s =+/−1 splits, thus from m _s =0 to m _s =+1 and from m _s = to m _s =−1 transitions to respond to microwave excitation of different frequencies. As long as the applied field is small or parallel to the symmetry axis of the NV center, the optical response remains unchanged and can continue to be used for reading. The optical polarization of the state m _s = 0 continues to serve as an ideal starting point _for experiments, and the first fluorescence intensity measured during an optical excitation pulse at a later time is It can be used to measure sub-level populations of 0, ie, can act as a sensitive longitudinal detection. The time interval between initial polarization and detection can be varied and used to perform sub-level spectroscopy between levels selected by the frequency of the microwave pulse. Note that an external electric field also splits the lower levels and can be used more than an external magnetic field.

All known microwave pulse sequences ranging from simple Hahn echoes, CPMG (Carr-Purcell-Meiboom-Gill) to MREV-8, search for the coherence time of the NV center, i.e. in millisecond time intervals. It can be used to determine field fluctuations as small as a few tenths of a milligauss. A prerequisite for these measurements is a long _T and freezing the movement of aptamers, proteins, and ions as well as the NV centers far from the surface (15-20 nm), i.e. freezing the liquid being tested. It requires that.

Furthermore, the change in distance between the spin label attached to the aptamer and the NV center induced by protein capture can be measured in DEER (Double Electron-Electron Resonance) experiments. Applying a second microwave excitation at a different frequency to flip the spins on the spin label while reversing the precession of the lower levels of the NV increases the difference in frequency of precession due to spin flipping. It can detect with precision and enables distance measurement with sub-nanometer precision of up to 20 nm. To perform DEER over the required distances, the relaxation times of the NV centers and spin labels on the aptamer need to be on the order of milliseconds, and it is again necessary to freeze the liquid being tested. .

As mentioned above, the lateral separation between NV centers may be large, eg, greater than 100 nm, to prevent significant interactions between NV centers. However, aptamers are small, on the order of a few nanometers, and we therefore distinguish between two different perceptions. In the first realization, aptamers are tightly bound to a surface such that any given NV center interacts with multiple randomly distributed aptamer sites within the range of interactions of the NV center. and thus measure the average interaction change across multiple sites.

In the second possibility, site-specific surface chemistry is used to ensure a 1:1 pairing between the aptamer site and the underlying NV center. This can be achieved by using surface chemistry influenced by the charge present in the NV center or by fluorescence of the NV center. This configuration has the advantage of providing nearly identical measurement sites providing single protein sensitivity and the ability to distinguish between precise target proteins and non-specific binders. This ability to gather statistics based on molecule-by-molecule yes or no decisions means that given the same number of NV centers, the second configuration can provide superior resolution and dynamic range.

One of the goals of this technology is to measure the concentration of multiple proteins simultaneously by using aptamers specific for each individual protein. There are at least two fundamentally different ways in which this can be achieved.

In the first possible detection paradigm, for any given protein target there are a large number of individual NV centers and aptamer sites, each measured by an independent detector. This efficiently performs a large number of single NV centered experiments. If there is still a 1:1 pairing between the NV center and the aptamer site, the number of bound proteins can be counted to yield a single number representing the concentration of the specific target protein.

In a second possible detection paradigm, multiple NV centers and aptamer sites for a given protein target are measured simultaneously by a single detector. Although this forgoes the advantage of molecule-by-molecule identification and requires creating a calibration curve to quantify the concentration of target protein, it may reduce the complexity of the required equipment.

In each case, ideally all NV centers are excited and detected simultaneously; however, it is important to use multiple measurement protocols to address the fact that different aptamers may have different responses. do not exclude.

In both cases, different parts of the diamond (or more generally crystal) surface are specialized to measure the concentration of different proteins. However, differences occur in the number of detectors required. For example, detecting the concentration of 10,000 proteins may require as many as ¹⁰⁸ detectors in the first case, but only 10,000 detectors in the second case. shall be.

Figures 8A-D represent different configurations for probing biochips based on the present technology. Identical NV centers that lie along the [111] direction close to the diamond surface terminating at (111) mean that all measured NV centers are equivalent and that most sensitive detection areas lie directly along the NV center. Although this is an ideal configuration, other constraints such as which orientations of diamond crystals can be easily produced may not allow this. Some device configurations tolerate this situation more than others.

FIG. 8A depicts a first exemplary configuration of an apparatus for detecting a target molecule, according to aspects of the present technology. In the simplest configuration, aptamer 301 is attached to the surface of diamond crystal 100 in close proximity to nitrogen-vacancy center 104 located at a predetermined depth below sensing surface 202 . The nature of the fluorescent emission light 418 induced or stimulated by the excitation light 208 is measured to detect the presence of the protein-aptamer complex 800. The presence of the protein induces a change in one or more properties of the nitrogen-vacancy center, which change is detected based on fluorescence emission from the nitrogen-vacancy center.

The configuration shown in FIG. 8A is all that is needed to perform all the optical T ₁ measurements described above. This imposes the least stringent condition on NV-centered equivalence. A (100) diamond surface with all eight different possible NV center orientations can be used. In this case, the axis of symmetry of the NV center is not perpendicular to this surface, which is not ideal, but all eight orientations are offset by the same amount, so the NV center functions equally well. One drawback is that the details of the dipole-dipole interaction minimize the magnetic interaction directly above the NV center, so that most sensitive interaction regions lie laterally from each NV center. be placed some distance apart in the direction. This does not apply to electrical interactions. This orientation is also not ideal for optical excitation and reading using light that is internally reflected within the diamond. However, this represents a good compromise between the two and allows the use of diamond surfaces, which are easier to manufacture.

FIG. 8B is another exemplary configuration of an apparatus for detecting a target molecule, according to aspects of the present technology. To the configuration represented in (A) is added a microwave source 806 in which microwave radiation 802 is directed onto the diamond surface by a microwave antenna 804. The microwave source was oriented to match the frequency of zero electrolytic splitting (2.89 GHz) and to ensure that the angle of the microwave magnetic field was the same for all NV centers in Figure 8B. In addition, the accuracy of the measurements may also be supported. The intensity of the fluorescent radiation 418 may be measured and the behavior of the resonance of the color center within the diamond may be identified based on the relationship between the measured intensity and the frequency of the microwave radiation 802.

FIG. 8C depicts yet another exemplary configuration of a device for detecting a target molecule, according to aspects of the present technology. To the configuration represented in (B) is added a magnetic field source 810 in which an external magnetic field 812 is imposed on the diamond surface. Adding an external static field H ₀ as described on FIG. 8C may be required for the measurement of phase coherence (T ₂ ).

FIG. 8D depicts yet another exemplary configuration of an apparatus for detecting a target molecule, according to aspects of the present technology. A second microwave source 808 that shares the same microwave antenna 804 as the first microwave source 806 is added to the configuration represented by (C). A second (independent frequency) microwave source, shown in Figure 8D, may be needed for spin labels to perform DEER experiments - all microwave magnetic fields are ideally for H ₀ Note that it is nearly vertical. At high magnetic fields, only NV centers parallel to the field can be optically read, so this configuration ideally uses a (111) surface with NV centers aligned along the [111] direction mentioned above. do. If all NV center orientations were equally populated, 75% of them would be unusable. Optical excitation and fluorescence emission are ideally approximately parallel to the NV axis.

In the case of non-equivalent NV centers (such as crystals where all eight inputs are terminated at (100) with different orientations), a possible solution is to The idea is to use only 1/8 of the NV center in the 200 nm and process all of these sequentially by applying different field orientations and microwave frequencies. Another potential solution is to use what is known as "field cycling" with resonant magnetic fields. In this case, a large field (e.g. more than two times larger than zero electrolytic splitting) can be applied along the [100] axis during microwave operation and switched to zero during optical polarization and detection.

FIG. 9 depicts yet another exemplary configuration of an apparatus 899 for detecting target molecules, in accordance with aspects of the present technology. More specifically, FIG. 9 is a schematic representation (not to scale) of a biochip integrated according to the invention. Although this figure represents one embodiment of a detection technique that utilizes spin labels, aptamers, and nitrogen-vacancy centers, other detection technique embodiments are possible. The biochip is composed of four layers: a capture layer 900, a diamond layer 902, a filter layer 904, and an integrated detection and processing layer 906. The capture layer 900 is comprised of a channel that directs a sample liquid containing a target molecule of interest 206 (e.g., a target protein 302) through the surface of a diamond layer onto which a SOMAmer 301 coupled to a magnetic spin label 300 is deposited. be done.

The features in the acquisition layer are areas of the sensing surface of the diamond layer onto which the same SOMAmer is deposited. Each mechanism contains one or more SOMAmers specific for separate protein analytes, x- This corresponds to the y coordinate (Fig. 9).

Each SOMAmer 301 or other capture reagent 200 may be a specific type or species of capture reagent configured to bind a specific type or species of target molecule 206. The capture reagent species may be attached to a functional group of the capture reagent. The capture layer 900 may include capture reagents 200 belonging to at least two different species, so that at least two target molecules 206 can be detected by the biochip device. In some embodiments, the capture layer 900 includes multiple species of capture reagents, such as hundreds, thousands, or more.

Some preferred embodiments of the invention do not include spin labels. Binding of a protein molecule to a SOMAmer molecule results in a change in the interaction 304 between the SOMAmer and the proximal nitrogen-vacancy center 104, and such change is detected via fluorescence emission 216. Surface chemistry and SOMAmers are optimized to yield large relative differences in interactions upon binding of protein targets to aptamers.

The diamond layer consists of high purity diamond fabricated by chemical vapor deposition into which a layer of NV centers 104 is embedded at a predetermined fixed distance 105 from the surface. In some examples, the predetermined fixed distance, which can also be expressed as depth, ranges from 5 to 20 nanometers, or from 15 to 20 nanometers. The NV centers within the diamond layer are separated by a distance greater than a predetermined fixed depth to ensure that interactions between them are small compared to interactions with molecules on the surface. The thickness of the layer at the NV center is small compared to a predetermined fixed depth. The distance between the nitrogen-vacancy center and the SOMAmer is selected such that a change in interaction upon binding of the protein to the SOMAmer is small enough to be possible and/or detectable. An external magnetic field 812 may be present. The diamond layer may include 100% ^12C diamond.

Excitation light 208 (provided by a light source such as a laser) stimulates fluorescence in the NV center. External microwaves 802 transition ground state electrons between these lower levels. The filter layer is constructed from a layered dielectric to form a dichroic bandpass filter 912, which is bonded to the diamond layer 902 on one side and the integrated detection and processing layer 906 on the other side. In addition to passing radiation from the NV center while blocking all other wavelengths, filter layer 904 also blocks radiation outside a narrow range of angles from the normal, thereby blocking adjacent NV Minimize crosstalk in center-to-center detection.

The filter layer is designed to be a narrow bandpass filter around the nitrogen-vacancy center emission wavelength (680 nm). Additionally, the filter layer blocks light outside the normal to narrow band angle to minimize crosstalk between adjacent features. The filter layer also acts as a mechanical bond between the diamond layer and the integrated detection and processing layer. An optically clear and mechanically rigorous adhesive can be used to bond the filter layer to the diamond layer on the top side and to the integrated detection and processing layer on the bottom side. Filter layers are constructed from dielectric layers that rely on the principle of wave interference to block light of specific wavelengths. The design and fabrication of filter layers is outside the scope of this technology.

The integrated detection and processing layer 906 is comprised of a CMOS avalanche photodetector 914 that drives a high speed electronic gate 916 that drives an electronic event counter 918 . Photodetector 914 may also be referred to as a detector assembly. In the present technique, a detector assembly may be configured to illuminate a color center with excitation light at one or more frequencies and to detect emission of electromagnetic radiation from the color center. Event counter output is collected for analysis. The designated coordinates 920 are chosen such that the x and y coordinates are in the plane of the surface of the upper diamond layer and the z coordinate is perpendicular to this surface. The range of wavelengths emitted by the light source (ie, the spectrum of excitation light 208) may or may not be the same as the range of wavelengths detected by photodetector 914. The range of wavelengths of the excitation light may partially overlap or not substantially overlap the range of wavelengths that photodetector 914 is configured to detect.

Integrated detection and processing layers may be designed for lithographic production and include both electronic components and connections between them. The integrated detection and processing layer may be fabricated using lithography and attached to the filter layer (or the diamond layer if the filter layer is excluded) using an optically clear adhesive. . The integrated detection and processing layer may consist of a photodetector layer, a gating layer, an AD (analog-to-digital) layer, and a bus for moving the accumulated data to the processor. Although FIG. 9 depicts these layers as being distributed in the z-direction, such an arrangement is not necessary.

The photodetector layer may include an array of avalanche photodiodes, each photodiode being specialized to and associated with a single feature on the capture layer in x and y coordinates. Each photodiode may be associated with a fast electron gate in a gating layer. Fluorescence changes from the NV center can be on the sub-microsecond scale, so it is necessary to collect light only in very short defined time frames to monitor these intensity changes. Each gate is in turn associated with an event counter in the event counter layer, which actually performs the photon counting. Data from each event counter is delivered to a processor for data analysis.

There are at least three example configurations in which the acquisition layer arrangement of FIG. 9 (ie, the area of the sensing surface of the diamond layer to which the SOMAmer is deposited) can be constructed, each offering different advantages. In the first example, a single SOMAmer is associated with a single NV center, which in turn is associated with a single photodetector. Such a setup offers advantages in terms of detection, but requires precise control of NV-centric or single SOMAmer placement. The main advantage of this form of construction is that it provides individual-based detection of molecules, which provides a third dimension of specificity (bond affinity and after washing). Furthermore, target molecules can be measured one by one, eliminating several calibration steps. The main drawback of this format is that a large number of such mechanisms are required to distinguish between different concentrations. Resolving orders of magnitude differences in concentration requires hundreds or thousands of such characterization mechanisms (based on a given level of resolution) for a single analyte, increasing instrument complexity. It is possible.

A second exemplary type of construct includes a homogeneous set of SOMAmers associated with a set of NV centers and thus a single photodetector. The random distribution of NV centers in the xy plane (defined in Fig. 9) and the random distribution of SOMAmers in the set of features results in an averaged signal at the photodetector, which is more It should be stable. A range of concentrations can also be quantified by multiple SOMAmers with a single photodetector. However, the use of such a collection instead of a single SOMAmer or NV center precludes the third dimension of specificity made possible by the first format, and the concentration of the target protein in the fluid depends on the NV Requires creation of a calibration curve to correlate center responses.

The third type of construct is a hybrid of the first and second types, involving multiple SOMAmers of the same type, associated with a single NV center, which in turn is associated with a single photodetector. Contains the set of Similar to the second format, a range of concentrations can be quantified with multiple SOMAmers on a single photodetector, which also results in an averaged signal. Use of a single NV center may result in a clearer signal. Randomly distributed NV centers can be optically located in the xy plane and then deposit the SOMAmer set at the same location.

FIG. 10 depicts steps performed in an exemplary method 1000 for detecting target molecules in a sample liquid and may not represent the complete process or all steps of the method. Although various steps of method 1000 are described below and depicted in FIG. 10, these steps need not all be performed, and may optionally be performed simultaneously or in a different order other than the order shown.

In step 1002, the method includes contacting a sample liquid with a capture reagent. The capture reagent is attached to a surface (eg, a crystalline film or substrate, or the surface of single crystal diamond) and is configured to bind to the desired target molecule. A capture reagent can be described as being captured by a surface, bound to a surface, attached to a surface, tethered to a surface, and/or immobilized to a surface. The target molecule can be any target molecule that has the property of binding the capture reagent, including, but not limited to, target molecules described in this disclosure such as proteins. Similarly, the capture reagent can be any suitable capture agent. This includes, but is not limited to, any of the capture reagents described in this disclosure, such as an aptamer, a nucleic acid molecule, or a nucleic acid molecule with at least one 5-position modified pyrimidine (such as a SOMAmer). . In some examples, at least two different 5-position modified pyrimidines are attached to the surface, including at least one 5-position modified uridine and at least one 5-position modified cytidine.

In step 1004, the method includes illuminating a color center located near the surface (eg, at a fixed depth within the crystal film) with excitation light. The excitation light is configured to direct fluorescence emission through the color center (eg, via selection of the wavelength or bandwidth of the center). In some examples, the color center is a nitrogen-vacancy center of a diamond crystal and the surface is a surface of the diamond crystal.

In step 1006, the method includes measuring the intensity of the fluorescent radiation using one or more detectors (e.g., a photodetector in the integrated detection and processing layer described above in connection with FIG. 9). including.

At step 1008, the method includes detecting a change in the intensity of the fluorescent emission. A change in intensity in response to binding of the target molecule to the capture reagent, and thus a change in emitted intensity, is indicative of the presence of the target molecule.

Optionally, in step 1010, the method includes irradiating the color center with microwave radiation at a frequency capable of inducing conversion of ground state electrons in the color center from the first substate to the second substate. may include. For example, the microwave radiation may include a resonant frequency that corresponds to the energy difference between a ground state zero spin substate and a ground state non-zero spin substate. Detecting the change in the intensity of the fluorescent emission in response to binding of the target molecule in step 1008 identifies the resonant behavior of the color center based on the relationship between the measured intensity of the fluorescent emission and the frequency of the microwave emission. The method may further include: For example, binding of a target molecule may induce a change in resonant frequency between the first substate and the second substate. Changes in the resonant frequency can be identified based on the intensity of the fluorescent radiation measured while varying the frequency of the microwave radiation.

FIG. 11 depicts steps performed in an exemplary method 1100 for measuring the concentration of a target molecule and may not represent the complete process or all steps of the method. Although various steps of method 1100 are described below and depicted in FIG. 11, these steps do not necessarily have to be performed, and may optionally be performed simultaneously or in a different order than the one shown.

In step 1102, the method includes exposing a fluid to a surface of a crystalline film (e.g., particularly a diamond film) to cause a capture reagent attached to the surface to bind to target molecules (e.g., particularly proteins) within the fluid. include. The crystalline film includes at least one color center, such as a nitrogen-vacancy center or other suitable defect.

At step 1104, the method includes irradiating the crystalline film with excitation light configured to induce fluorescence emission from at least one color center within the film. Optionally, step 1104 includes irradiating the color center with microwave radiation having a frequency capable of inducing a conversion of ground state electrons at the color center from the first substate to the second substate. .

At step 1106, the method includes detecting a change in the properties of the color center based on the fluorescence emission. A change in properties at the color center occurs upon binding of the target molecule by the capture reagent. In some examples, the capture reagent includes a magnetic spin label such that binding between the target molecule and the capture reagent alters the magnetic field at the color center. Changes in properties at color centers can be brought about, at least in part, by changes in the magnetic field.

At step 1108, the method includes determining a concentration of target molecules within the fluid based on the detected property change in the color center. For example, the detected change signature may represent the number or approximate number of target molecules bound by the capture reagent to the surface of the membrane, and the concentration of target molecules within the fluid may be determined from the number of target molecules and the volume of the fluid. It can be estimated.

FIG. 12 depicts steps performed in an exemplary method 1200 for detecting a target molecule and may not represent the complete process or all steps of the method. Although various steps of method 1200 are described below and depicted in FIG. 12, these steps do not necessarily have to be performed, and may optionally be performed simultaneously or in a different order than the order shown.

In step 1202, the method includes exposing a crystalline substrate (e.g., a diamond film) to a sample liquid such that a capture reagent attached to a surface of the substrate can bind target molecules predicted to be present in the sample liquid. include. The crystalline substrate includes at least one color center, such as a nitrogen-vacancy center in diamond. The target molecule can be a protein or any other target molecule described elsewhere in this disclosure. Similarly, the capture reagent can be any molecule suitable for specifically binding the desired target molecule, and can include aptamers, SOMAmers, and the like. In some examples, the capture reagent is an aptamer that includes at least two distinctly different 5-position modified pyrimidines.

At step 1204, the method includes irradiating the crystal substrate with electromagnetic radiation. The electromagnetic radiation is configured to stimulate or induce fluorescence due to color centers within the crystal substrate. Characteristics of the electromagnetic radiation, including center frequency, bandwidth, intensity, pulse energy, pulse duration, and/or polarization, can be designed to, at least in part, determine the characteristics of the emitted fluorescence.

At step 1206, the method includes detecting fluorescence emitted by the color center, eg, with one or more photodetectors.

At step 1208, the method includes identifying a change in the properties of one or more color centers resulting from binding one or more target molecules from the sample liquid to a capture reagent. For example, identifying this change may include identifying a change in the spectrum of radiation emitted by the color center, such as detecting fluorescent radiation emitted by the color center, the intensity of the emitted fluorescence; It may include identifying temporal variations, or changes in the spectrum.

FIG. 13 depicts steps of an exemplary method 1300 for manufacturing a device for detecting the presence of protein molecules in a sample liquid, and may not represent the complete process or all steps of the method. Although various steps of method 1300 are described below and depicted in FIG. 13, these steps do not necessarily have to be performed, and may optionally be performed simultaneously or in a different order than the order shown. Aspects of method 1300 can be used, for example, to fabricate the integrated biochip depicted in FIG. 9.

At step 1302, the method includes fabricating a crystalline film, such as by chemical vapor deposition. For example, ¹² C-rich diamond films can be produced by plasma-enhanced chemical vapor deposition (CVD) of a mixture of methane and hydrogen gases on a diamond substrate.

At step 1304, the method includes embedding replacement atoms in the crystalline film. Substitute atoms are suitable for placement with vacancies in the crystal film to form color centers.

At 1306, vacancies are created in the crystalline film, such as by bombarding the film with an ion beam and/or an electron beam.

At step 1308, the method includes creating color centers in the crystalline film by co-locating at least some of the vacancies and at least some of the replacement atoms. Co-locating substitution atoms and vacancies may include annealing the film, eg, at high temperature or by any other suitable process.

At step 1310, the method includes depositing a capture reagent on a first surface on the crystalline film. Step 1310 may include attaching a capture reagent to some regions of the surface of the crystalline membrane and passivating regions of the surface not bound to the capture reagent against non-specific binding by unwanted proteins. . Attaching the capture reagent and passivating other areas of the surface involves attaching the passivating molecule ( For example, this can be achieved by bonding a hydrophilic polymer). For example, a portion of the passivated molecule may have a first end configured to bind to a surface and a second end having an active group configured to bind to DNA or other target molecules. may have a section.

At step 1312, the method includes attaching a photodetector to the second surface of the crystalline film such that the photodetector can detect emission from the color center. The photodetector may be fabricated using lithography and bonded to the crystal film using an optically transparent and mechanically stringent adhesive. In some examples, a filter layer (eg, filter layer 904) is included between the photodetector and the membrane.

A typical detection scheme for determining the concentration of a target molecule in a sample liquid is to measure the intensity of the fluorescence emission due to at least one color center, the fluorescence emission in response to the binding of at least one target molecule to a capture reagent. and determining the concentration of the target molecule in the sample liquid based on the identified change. For example, the excitation and emission aspects of the nitrogen vacancy center depicted in FIG. 7 may be included in an exemplary method in which the relaxation time T ₁ is monitored entirely by optical methods. T ₁ is measured by polarizing the ground spin state to a level below m _s =0 by illuminating the NV center with excitation light. As shown in Fig. 4, the fraction of electrons excited from the lower level with m _s = ±1 returns to the ground state via a dark transition, which then undergoes a transition to the lower level with m _s =0. Provide maximum strength. T ₁ is determined by periodically monitoring the fluorescence intensity after excitation-mediated polarization is discontinued and seeing how quickly the ground state returns to a fully unpolarized state. An exemplary method for measuring protein in urine includes the following steps, which may be performed simultaneously or in a different order than the order shown.
1. Prepare multiple calibration samples of urine containing known concentrations of the protein of interest.
2. These calibration samples are flowed over the sensing surface of the diamond layer, providing sufficient opportunity for proteins to bind to the attached SOMAmer, and optionally recirculating the sample liquid.
3. A wash solution is flowed over the fluid-contacting surface of the diamond layer, providing the wash solution with sufficient opportunity to remove molecules that bind in a non-specific manner to SOMAmer, and optionally the wash solution is recirculated.
4. The NV center is irradiated with a series of pulses of excitation light, each pulse having a duration long enough to result in complete polarization of the NV center to the m _s =0 state. The time interval τ _i between these excitation pulses varies.
5. The fluorescence intensity immediately after the firing of each excitation pulse is measured, as well as the reference intensity taken towards the end of each excitation pulse when the spin state is fully polarized to a level below m _s =0.
6. Calculate the normalized intensity by dividing the fluorescence intensity at the start of each excitation pulse by the corresponding reference intensity.
7. A response plot for each calibration sample is created by plotting the reference intensity of each pulse _{p i} against the interval time t _i immediately preceding pulse p i.
8. The relaxation time T ₁ for each calibration sample is found by fitting a decaying exponential function of form normalized intensity = I ₀ *exp(-τ/T ₁ ).
9. Create a calibration curve of protein concentration vs. _T1 .
10. To generate a response plot, steps (2)-(8) are repeated using the sample of interest instead of the calibration sample.
11. Apply the calibration curve of step 9 to determine the protein concentration for each sample of interest.

Further methods may include the system shown in FIG. 8, for example. Although optical detection of spin states is preferred, the addition of microwave excitation and the introduction of static fields extends all conventional EPR-derived techniques for manipulating spin states. Examples of these (eg Hahn echo, DEER, etc.) are discussed above. There are many variations on the above method, with the addition of an additional spinning step via techniques well known in the art, which may be selected to optimize detection of bound analyte.

Although the given examples mainly focus on urine or blood (serum or crystals) as sample fluids, biochips according to the present technology can work with various biological fluids or solutions derived from these, e.g. by dilution. It is possible. These include, but are not limited to, blood, plasma, serum, urine, semen, saliva, meningeal fluid, amniotic fluid, glandular fluid, lymph, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, and white blood cells. , peripheral blood mononuclear cells, sputum, breath, cells, cell extracts, feces, tissues, tissue extracts, tissue biopsies, and cerebrospinal fluid.

Exemplary Combinations and Additional Examples This section describes further aspects and mechanisms of proteomic assays, presented without limitation as a series of paragraphs, some of which may improve clarity and efficiency. May be presented alphabetically for gender. Each of these paragraphs may be combined in any suitable manner with one or more other paragraphs and/or the disclosure elsewhere in this application, including any material incorporated by reference. Some of the following paragraphs explicitly refer to and further limit other paragraphs and provide examples, but are not limiting, of some suitable combinations.

A. An apparatus for the detection of one or more species of target molecules in a fluid, the apparatus comprising:
a) a solid support having a surface;
b) a capture reagent of one or more species attached to the surface,
c) capture reagents, each of which selectively binds to a specific species of target molecule;
d) a color center located near the surface of the solid support;
e) A device comprising a detector for detecting a change in the properties of a color center upon binding of a protein molecule to said capture reagent.

A1. The device of paragraph A, wherein the fluid comprises a biological fluid and the target molecule is a protein.

A2. The device according to paragraph A1, wherein the capture reagent is an aptamer.

A3. The device according to paragraph A2, wherein the aptamer is a nucleic acid molecule.

A4. The device according to paragraph A3, wherein at least a portion of the nucleic acid molecule has at least one pyrimidine modified at the 5-position.

A5. The device according to paragraph A4, wherein the solid support is a diamond crystal and the color center is a nitrogen-vacancy center.

A6. The apparatus of paragraph A5, wherein the detector is an optical system that illuminates the nitrogen-vacancy center with radiation of a first range of wavelengths and detects a second range of wavelengths.

A7. The apparatus according to paragraph A6, wherein the light source is a pulsed light source.

A8. The apparatus of paragraph A7, wherein the change in the nature of the nitrogen-vacancy center is a change in the magnetic resonance nature of the nitrogen-vacancy center.

A9. The apparatus of paragraph A8, further comprising a magnetic field source for providing a magnetic field.

A10. The apparatus of paragraph A9, further comprising a microwave source for providing microwave radiation.

A11. The apparatus of paragraph A10, wherein the microwave source is tuned to a frequency that resonates with a lower level of the electron ground state of the nitrogen-vacancy center.

A12. The apparatus of paragraph A11, wherein the microwave source is a pulsed source.

A13. The device according to paragraph A5, wherein the surface of the solid support is a {111} surface of single crystal diamond.

A14. The device according to paragraph A5, wherein the surface of the solid support is a {100} surface of single crystal diamond.

The apparatus of paragraph A5, wherein the nitrogen-vacancy center is located within the surface of a 25 nanometer diamond crystal.

A16. The apparatus of paragraph A7, wherein the nucleic acid molecule further comprises a spin label.

A17. The apparatus according to paragraph A12, wherein the nucleic acid molecule further comprises a spin label.

B. An apparatus for simultaneously quantifying single or multiple analytes in a sample liquid, the apparatus comprising:
a) a thin crystalline layer with one side in contact with the sample liquid and containing a color center defect 5 to 25 nanometers from the surface in contact with the fluid;
b) as described in (a), such that binding of the analyte to the binding agent influences a local magnetic field external to the color center defect, thereby detectably altering the behavior of said color center defect; a scavenger deposited on the fluid-contacting surface of the crystalline layer in the immediate vicinity of the color-centered defect;
c) An apparatus comprising means for detecting a change in the behavior of said color center defect.

B1. The change in behavior of the color center defect includes a change in fluorescence, and the means for detecting the change in fluorescence comprises:
a) an optical filter layer coupled to a non-contact surface of the crystal layer for passing fluorescent radiation from the color center defect while excluding excitation light or other light;
b) a detection layer coupled to an optical filter layer on the side opposite said crystal layer for the capture and quantification of fluorescence emitted by color center defects, including intensity;
c) An apparatus according to paragraph B, comprising means for introducing excitation light into the crystal layer such that the color center defects are stimulated to emit fluorescence.

B2. Means for introducing said excitation light into a crystalline layer directs the introduction of said light through an edge of said crystalline layer such that said excitation light travels in a direction parallel to the fluid contacting surface of said crystalline layer. Apparatus according to paragraph B1 for carrying out.

B3. The change in behavior of the color center defect includes a change in fluorescence, and the means for detecting the change in fluorescence comprises:
a) an optical waveguide, optionally including an optical filter, coupled to a non-contacting surface of the crystal layer, for passing fluorescent radiation from the color center defect while excluding excitation light or other light;
b) a detection layer located on the opposite side of the optical waveguide from said crystalline layer for the capture and quantification of the fluorescence emitted by the color-centered defects, including the intensity;
c) The apparatus according to paragraph B, comprising means for introducing excitation light into the crystal layer such that the color center defects are stimulated to emit fluorescence.

B4. the change in behavior of the color center defect comprises a change in an electric or magnetic field local to the color center defect, and the means for detecting the change in the electric or magnetic field comprises an electronic or optoelectronic detector; Apparatus according to paragraph B.

B5. Apparatus according to paragraph B1, further comprising means for introducing microwave radiation into the crystal layer for influencing and/or monitoring the resonant behavior of the color center defects.

B6. Apparatus according to paragraph B5, comprising means for imposing a constant or varying magnetic field over the region containing the color center defect to modify the resonance behavior.

B7. The apparatus of paragraph B5, wherein the thin crystalline layer is a diamond film and the color center defect is a nitrogen-vacancy center.

B8. The device of paragraph B7, wherein the device can be refurbished for multiple uses.

B9. A binding agent specific for the analyte is attached to a magnetic spin label for increasing the action upon binding of the analyte on a local magnetic field external to the color center defect, thereby The apparatus of paragraph B8, which detectably alters the behavior of the central defect.

B10. The device according to paragraph B9, wherein the analyte-specific binding agent is an aptamer or a SOMAmer.

B11. The apparatus of paragraph B9, wherein the optical filter layer is comprised of a dielectric film layered to form a dichroic bandpass filter.

B12. The detection layer comprises a CMOS avalanche photodetector in immediate contact with the optical filter layer, the signal from the photodetector passes through a fast electronic gate, and the signal passing through the fast gate The apparatus of paragraph B9, wherein the device is passed to an event counter and the resulting data collected by the event counter is delivered to a processor for analysis.

B13. The collection of tethered SOMAmers is identical in their specificity for a specific analyte and is measured with the collection of nitrogen-vacancy centers that collectively emit fluorescence that is captured by individual photodetectors. The apparatus according to paragraph B9, interacting in a possible manner.

B14. The device according to paragraph B13, wherein the collection of SOMAmers consists of a single molecule, and the collection of nitrogen-vacancy centers consists of a single center.

B15. The device of paragraph B13, wherein the collection of SOMAmers consists of molecules that are identically specific for the same analyte, and the collection of nitrogen-vacancy centers consists of a single center.

B16. The device of paragraph B13, wherein there is a plurality of said collections of tethered SOMAmers, each said collection being specific for a different analyte.

B17. The apparatus according to paragraph B16, wherein the number of the set of tethered SOMAmers is between 100 and 10,000.

B18. means for directing the fluid to the fluid-contacting surface of the diamond layer, including means for recirculating the fluid as necessary;
a) sample liquid;
b) a cleaning solution used to remove molecules that bind in a non-specific manner to the fluid contacting surface of the SOMAmer or the diamond layer;
c) binding both specifically and non-specifically to the fluid-contacting surface of the SOMAmer or the diamond layer without causing damage or modification to the fluid-contacting surface of the SOMAmer or the diamond layer; The apparatus of paragraph B13, comprising a regeneration liquid used to remove molecules that cause

B19. The device of paragraph B18, wherein the sample fluid is a biological fluid derived from a human subject.

B20. The device according to paragraph B19, wherein the biological fluid is urine.

B21. According to paragraph B20, the device is designed to be contained within a toilet, urinal, or urine container, thereby allowing capture and analysis of urine from the toilet, urinal, or urine container. Device.

B22. The device of paragraph B21, wherein the data generated by the device is compared to a proteomics database for the diagnosis of possible disease states.

B23. How the analyte concentration is described:
a) preparing a plurality of calibration samples of a fluid containing known concentrations of the analyte;
b) directing the calibration sample to a fluid-contacting surface of the diamond layer, optionally including recirculation of the calibration sample liquid, and providing sufficient opportunity for binding to the tethered SOMAmer for the analysis; to provide something;
c) directing the cleaning solution onto the fluid-contacting surface of the diamond layer, giving the cleaning solution sufficient opportunity to remove molecules that bind to the SOMAmer in a non-specific manner; directing the opportunity, optionally including recirculation of the cleaning solution;
d) irradiating said nitrogen-vacancy center with excitation light, said light comprising a frequency that induces fluorescence emission of said nitrogen-vacancy center, and measuring said fluorescence emission intensity; to do and
e) irradiating the nitrogen-vacancy center with microwave radiation, the frequency of the microwave radiation changing the nitrogen-vacancy center from a spin state of 0 to a spin state of ±1; irradiating, varying over a range predicted to include a resonant frequency that induces conversion of ground state electrons;
f) creating a plot of the fluorescence radiation intensity versus the frequency of the microwave radiation for each of the calibration samples;
g) creating a calibration curve of some selected features of the plot of said calibration sample from analyte concentration versus (f);
h) repeating steps (b) to (f) using the sample of interest instead of the calibration sample to produce a plot as in (f);
The apparatus of paragraph B18, wherein: i) the calibration curve of step (f) is applied to determine the concentration of each analyte of the sample of interest measured in the sample liquid.

B24. The apparatus and method of paragraph B13, wherein the selected feature of the plot is the resonant microwave frequency, determined by locating the most extreme local minimum.

B25. 24. The apparatus and method of paragraph 23, wherein selected features of the plot represent the width of the resonant inversion peak in some form, such as full width at half maximum (FWHM), or coefficient of variation (CV).

B26. How the analyte concentration is described:
a) preparing a plurality of calibration samples of a fluid containing known concentrations of the analyte;
b) directing the calibration sample to a fluid-contacting surface of the diamond layer, optionally including recirculation of the calibration sample liquid, and providing sufficient opportunity for binding to the tethered SOMAmer for the analysis; giving things and
c) directing the cleaning solution onto the fluid-contacting surface of the diamond layer, giving the cleaning solution sufficient opportunity to remove molecules that bind in a non-specific manner to the SOMAmer; the opportunity to direct, optionally including recirculation of the cleaning fluid;
d) irradiating said nitrogen-vacancy center with a series of square-wave pulses p _i of excitation light, said light comprising a frequency that induces fluorescence emission of said nitrogen-vacancy center, each pulse each of which is long enough to bring about complete polarization of the nitrogen-vacancy center to the zero spin state, further varying the spacing τ _i between these excitation pulses p _i ;
e) measuring the fluorescence intensity immediately after the activation of each of said excitation pulses and a reference intensity taken for the end of each of said excitation pulses, said reference intensity being such that the to be measured, to be measured if it should be polarized;
f) calculating a normalized intensity by dividing the starting fluorescence intensity of each excitation pulse p _i by the corresponding reference intensity;
g) creating a response plot for each calibration sample by plotting the reference intensity of each pulse _{p i against} the interval time t _{i immediately preceding pulse p i} ;
h) Formula y=I ₀ * exp (-τ/T1) (where y is a dependent variable consisting of a set of normalized intensities of each of the above pulses p _i , and an independent variable τ is a finding the relaxation time T ₁ of each of said calibration samples by fitting a decaying exponential function of p _i ), which is the set of interval times preceding each of p i;
i) creating a calibration curve of known analyte concentration in said calibration sample versus relaxation time T ₁ from step (h);
j) repeating steps (b) to (h) using the sample of interest instead of the calibration sample to produce a plot as in (g), and relaxing as in step (h); finding time and
k) Apparatus according to paragraph B18, measured in said sample liquid according to applying the calibration curve of step (i) to determine the concentration of the analyte of each sample of interest.

C. A device for detecting a target molecule, the device comprising:
a surface calibrated to contact the fluid;
a plurality of capture reagents attached to the surface, each configured to bind a target molecule;
a plurality of color centers located proximal to the surface;
An apparatus comprising at least one detector configured to detect a change in at least one property of a color center in response to binding of a target molecule to one of the capture reagents.

C1. The device according to paragraph C, wherein the target molecule is a protein and the capture reagent is an aptamer.

C2. The device according to paragraph C1, wherein the aptamer is a nucleic acid molecule.

C3. The apparatus of paragraph C2, wherein the nucleic acid molecule has at least one pyrimidine modified at the 5-position.

C4. The apparatus of paragraph C, wherein the surface is a surface of a diamond crystal, and the color center is a nitrogen-vacancy center of the diamond crystal;

C5. The apparatus according to paragraph C4, wherein the diamond crystal is single crystal diamond.

C6. further comprising a light source configured to illuminate the nitrogen-vacancy center with radiation having a wavelength in a first range, and the detector configured to detect radiation having a wavelength in a second range. , the apparatus according to paragraph C4.

C7. Apparatus according to paragraph C4, wherein the property is related to nitrogen-vacancy centered magnetic resonance.

C8. The apparatus of paragraph C4, further comprising a microwave source configured to provide microwave radiation having a frequency in a range that includes a resonant frequency below the ground state of a nitrogen-vacancy centered electron.

C9. The device of paragraph C, wherein the capture reagents include reagents belonging to multiple capture species, each capture species configured to bind to a target molecule of a particular target species.

C10. A device for measuring the concentration of a target molecule, the device comprising:
a crystalline film containing at least one color center;
a plurality of capture reagents attached to the surface of the crystalline membrane and configured to bind to the target molecules;
An apparatus comprising a detector assembly configured to illuminate the color center with excitation light and detect emission of electromagnetic radiation from the color center.

C11. The apparatus of paragraph C10, wherein the capture reagents include magnetic spin labels, and the magnetic field of the color center changes in response to binding of a target molecule to one of the capture reagents.

C12. The detector assembly is configured to illuminate the color center with microwave radiation having a frequency capable of inducing conversion of ground state electrons at the color center from a first substate to a second substate. The apparatus according to paragraph C11.

C13. The apparatus according to paragraph C10, wherein the crystalline film is a diamond film and the color center is a nitrogen-vacancy center.

C14. Detection in the emission of electromagnetic radiation from the color center by binding of one of the capture reagents to one of the target molecules changes the interaction between the spin label of the capture reagent and the color center. Apparatus according to paragraph C10, which brings about possible changes.

C15. A device for detecting a target molecule, the device comprising:
a plurality of capture reagents attached to the surface of the crystal substrate and configured to capture target molecules from the sample liquid;
a plurality of color centers disposed at a fixed distance from said capture reagent, said fixed distance being such that at least one property of said color center is responsive to capture of one of said target molecules by one of said capture reagents; With multiple color centers small enough to vary,
An apparatus comprising a detector configured to detect a change in at least one property of the color center.

C16. The device according to paragraph C15, wherein the capture reagent is an aptamer.

C17. The device of paragraph C16, wherein the capture reagent is an oligonucleotide.

C18. The device of paragraph C16, wherein the capture reagent is a nucleic acid molecule having a modified pyrimidine at least at the 5-position.

C19. The apparatus according to paragraph C18, wherein the crystal substrate is a diamond film and the color center is a nitrogen-vacancy center.

D0. A method for detecting a target molecule in a sample liquid, the method comprising:
contacting the sample liquid with a capture reagent attached to the surface and configured to bind to the target molecule;
irradiating the color center located proximal to the surface with excitation light configured to induce fluorescence emission by the color center;
measuring the intensity of said fluorescent radiation using one or more detectors;
A method comprising detecting a change in the intensity of said fluorescent emission in response to binding of said target molecule to said capture reagent.

D1. further comprising irradiating the color center with microwave radiation at a frequency capable of inducing conversion of ground state electrons at the color center from a first substate to a second substate;
detecting a change in the intensity of the fluorescent radiation in response to binding of the target molecule to the capture reagent at the color center based on the relationship between the measured intensity of the fluorescent radiation and the frequency of the microwave radiation; identifying the resonant behavior of the
The method described in D0.

D2. The method according to paragraph D0 or D1, wherein the target molecule is a protein.

D3. The method according to any one of paragraphs D0-D2, wherein the capture reagent is an aptamer.

D4. The method of any one of paragraphs D0-D3, wherein the capture reagent is a nucleic acid molecule having a pyrimidine modified at least at the 5-position.

D5. The method according to any one of paragraphs D0 to D4, wherein the surface is a surface of a diamond crystal, and the color center is a nitrogen-vacancy center of the diamond crystal.

D6. The capture reagent is an aptamer comprising at least one first pyrimidine modified at the 5-position and at least one second pyrimidine modified at the 5-position, the first pyrimidine modified at the 5-position and The second pyrimidine modified at the 5-position is a pyrimidine modified at a different 5-position,
The first pyrimidine modified at the 5-position is uridine modified at the 5-position, and the second pyrimidine modified at the 5-position is a cytidine modified at the 5-position, or 1, the pyrimidine modified at the 5-position is cytidine modified at the 5-position, and the second pyrimidine modified at the 5-position is uridine modified at the 5-position,
The method according to any one of paragraphs D0 to D5.

E0. A method for measuring the concentration of a target molecule, the method comprising:
exposing a fluid to a surface of the crystalline membrane to cause a capture reagent attached to the surface to bind to a target molecule within the fluid;
irradiating the film with excitation light configured to induce fluorescence emission by at least one color center in the crystalline film;
detecting, based on the fluorescence emission, a change in the properties of a color center in response to binding between the target molecule and the capture reagent;
A method comprising determining a concentration of a target molecule in the fluid based on the detected change.

E1. The method according to paragraph E0, wherein the target molecule is a protein.

E2. The method of paragraph E1 or E2, wherein the capture reagent comprises a magnetic spin label and the magnetic field of the at least one color center changes in response to binding between the target molecule and the capture reagent.

E3. Irradiating the membrane with the excitation light stimulates the color center with microwave radiation having a frequency capable of inducing a conversion of ground state electrons at the color center from a first substate to a second substate. The method of any one of paragraphs E0-E2, comprising irradiating.

E4. The method according to any one of paragraphs E0 to E3, wherein the color center is a nitrogen vacancy center.

E5. The method according to any one of paragraphs E0 to E4, wherein the crystalline film is a diamond film.

F0. A method for detecting a target molecule, the method comprising:
exposing a crystalline substrate to the sample liquid such that a plurality of capture reagents attached to the surface of the crystalline substrate bind to target molecules in the sample liquid;
A method comprising identifying a change in the properties of one or more color centers within the crystalline substrate in response to binding of the target molecule to the capture reagent.

F1. The method of paragraph F0, wherein the crystal substrate is a diamond film, and the color center is a nitrogen-vacancy center within the diamond film.

F2. Paragraph F0 or F1 further comprising irradiating the crystalline substrate with electromagnetic radiation, and identifying a change in the properties of one or more color centers comprises detecting fluorescent radiation emitted by the color center. The method described in.

F3. The method of any one of paragraphs F0-F2, wherein the capture reagent is an aptamer comprising at least two distinctly different 5-position modified pyrimidines.

F4. The method according to any one of paragraphs F0 to F3, wherein the target molecule is a protein.

G0. A method for manufacturing a device for detecting the presence of protein molecules in a sample liquid, the method comprising:
producing a crystalline film;
embedding a plurality of replacement atoms in the crystal film;
Creating a plurality of pores in the crystal film;
creating color centers in the crystalline film by arranging at least some substitution atoms with at least some vacancies;
attaching a plurality of capture reagents to the first surface of the crystalline film;
A method comprising depositing a layer of photodetector on a second surface of the crystalline film.

G1. The method of paragraph G0, wherein the crystalline film is fabricated using chemical vapor deposition.

G2. The method according to paragraph G0 or G1, wherein the holes are created using an electron beam.

G3. The method of any one of paragraphs G0-G2, wherein the substitution atoms are placed with the vacancies by annealing the crystalline film at high temperatures.

G4. Depositing the photodetector layer includes fabricating the photodetector layer using lithography and attaching the photodetector layer to the second surface of the crystalline film using an optically transparent adhesive. The method of any one of paragraphs G0-G3, comprising adhering the layers of the photodetector.

Claims (20)

A method for measuring the concentration of a label-free target molecule in a sample liquid, the method comprising:
exposing the substrate to the sample liquid such that a plurality of capture reagents attached to the surface of the substrate contact the sample liquid, wherein the plurality of capture reagents contact the first label-free molecule; a first capture reagent configured to bind and a second capture reagent configured to bind a second label-free molecule different from the first label-free molecule; process and
a change in the properties of the at least one color center in response to binding between the first label-free molecule and the first capture reagent based on the emission of electromagnetic radiation from the at least one color center in the substrate; a step of detecting;
Measuring the concentration of the first label-free molecule in the sample liquid based on the detected change;
including methods. 2. The method of claim 1, wherein the first and second label-free molecules are proteins and the first and second capture reagents are aptamers. 3. The method of claim 2, wherein each aptamer has at least one pyrimidine modified at the 5-position. 2. The method of claim 1, wherein the substrate comprises a diamond crystal and the at least one color center is a nitrogen-vacancy center of the diamond crystal. 5. The method of claim 4, wherein the property is related to magnetic resonance of the color center. The method further comprises irradiating the at least one color center with radiation having a first wavelength range configured to induce the emission of electromagnetic radiation, the emitted electromagnetic radiation 2. The method of claim 1, wherein the second wavelength range is not identical to the wavelength range of the second wavelength range. 7. The method of claim 6, further comprising irradiating the at least one color center with microwave radiation having a frequency range that includes a resonant frequency below a ground state of an electron of the at least one color center. 2. The method of claim 1, wherein each of the first capture reagents comprises a magnetic spin label. The surface includes an array of separate regions each containing only one of the first capture reagents, and the at least one color center includes a plurality of color centers each associated with one of the first capture reagents. 2. The method of claim 1, comprising: A method for detecting a label-free target analyte in a sample liquid, the method comprising:
exposing at least two capture reagents attached to a surface to a sample liquid, each capture reagent configured to bind a different label-free target analyte, the surface having a plurality of colored centers; the exposing step being arranged in close proximity;
detecting a change in the properties of a first of the plurality of color centers in response to binding of one of the different label-free target analytes to one of the corresponding capture reagents;
including methods. 11. The method of claim 10, wherein detecting the change includes detecting the emission of electromagnetic radiation from the first color center with a photodetector. 12. The method of claim 11, wherein the photodetector is configured to detect emission from only the first color center of the plurality of color centers. 12. The method of claim 11, wherein the capture reagent is an aptamer. 14. The method of claim 13, wherein each of the aptamers comprises a magnetic spin label. A method for detecting a label-free target molecule, the method comprising:
capturing a first label-free target molecule from a sample liquid by a first capture reagent of a plurality of capture reagents attached to a surface, wherein the first capture reagent is attached to the first label. The plurality of capture reagents belong to a first species configured to bind a free target molecule, and the plurality of capture reagents belong to a second species configured to bind a second label-free target molecule. said capturing step further comprising a capture reagent of;
detecting capture of the first label-free target molecule by the first capture reagent based on the emission of a first electromagnetic radiation from a color center located adjacent to the surface;
including methods. 16. The method of claim 15, wherein the first and second label-free molecules are proteins and the first and second capture reagents are aptamers. 17. The method of claim 16, wherein the first capture reagent has a modified pyrimidine base at the 5-position. 16. The method of claim 15, further comprising exciting the color center with a second electromagnetic radiation causing emission of first electromagnetic radiation from the color center. 16. The method of claim 15, wherein detecting capture of the first label-free target molecule comprises detecting the first electromagnetic radiation with a photodetector. 16. The method of claim 15, wherein the first capture reagent comprises a magnetic spin label.

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